The osteoblast-specific transcription factor Cbfa1 contributes to the expression of osteoprotegerin, a potent inhibitor of osteoclast differentiationand function

Bone formation and resorption are tightly coupled under normal conditions, and the interaction of osteoclast precursors with cells of the osteoblast l ineage is a prerequisite for osteoclast formation. Cbfa1 is an osteoblast-s pecific transcription factor that is essential for osteoblast differentiati on and bone formation. At present, it is not known whether Cbfa1 regulates any of the osteoblast-derived factors involved in the bone resorption pathw ay. Osteoprotegerin (OPG) is an osteoblast-secreted glycoprotein that funct ions as a potent inhibitor of osteoclast differentiation and bone resorptio n, Cloning and computer analysis of a 5,9-kilobase human OPG promoter seque nce revealed the presence of 12 putative Cbfa1 binding elements (osteoblast -specific element 2 (OSE2)), suggesting a possible regulation of OPG by Cbf a1, We cloned the promoter upstream of the beta-galactosidase reporter gene (pOPGEi.Bpgal) and evaluated whether Cbfa1 could regulate its expression i n transient transfection assays. The 5,9-kilobase promoter directed increas ed levels of reporter gene expression, reminiscent of OPG protein levels in osteoblastic cell lines (BALC and U2OS) as compared with the nonosteoblast ic cell line COS1, Cotransfection of a Cbfa1 expression construct along wit h pOPG5.9 beta gal reporter construct led to 39-, 7-, and 16-fold increases in beta-galactosidase activity in COS1, BALC, and U2OS cells, respectively . Removal of all the putative OSE2 elements led to an almost complete loss of transactivation, Mutational analysis demonstrated that the proximal OSE, element contributes to a majority of the effects of Cbfa1, and Cbfa1 bound to the proximal element in a sequence-specific manner. Further, overexpres sion of Cbfa1 led to a 54% increase in OPG protein levels in U2OS cells. Th ese results indicate that Cbfa1 regulates the expression of OPG, thereby fu rther contributing to a molecular link between bone formation and resorptio n.